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laboratory science

Blue light-absorbing intraocular lens and retinal


pigment epithelium protection in vitro
Janet R. Sparrow, PhD, Ashley S. Miller, Jilin Zhou, MD

Purpose: To compare the Alcon AcrySof威 Natural (SN60AT) and AcrySof


(SA60AT), the AMO Sensar威 (AR40e) and ClariFlex威, and the Pfizer CeeOn威 Edge
911A intraocular lenses (IOLs) as to their ability to protect retinal pigment epithe-
lial (RPE) cells from light damage mediated by the lipofuscin fluorophore A2E.
Setting: Department of Ophthalmology, Columbia University, New York, New
York, USA.
Methods: Cultured human RPE cells (ARPE-19 cell line) that had accumulated
A2E were exposed to blue (430 nm ⫾ 30), green (550 ⫾ 10 nm), or white (390 to
750 nm) light with and without an IOL in the light path.
Results: The blue light-absorbing AcrySof Natural IOL was associated with sig-
nificant reduction (78% to 82%; P⬍.01) in the death of A2E-laden RPE that were
exposed to blue, white, and green light. The decrease in the incidence of cell
death was greater in magnitude than would be expected from the amount of light
that was absorbed by the IOL. The considerably smaller declines in cell death ob-
served with the AcrySof, Sensar, ClariFlex, and CeeOn Edge IOLs were likely due
to nonspecific reductions in light transmittance.
Conclusions: By absorbing blue light, the AcrySof Natural IOL shields RPE cells
that have accumulated the aging lipofuscin fluorophore A2E from the damaging ef-
fects of light. A long-term population-based clinical trial would determine whether
a blue light-absorbing IOL can reduce the risk for or progression of age-related
macular degeneration.
J Cataract Refract Surg 2004; 30:873–878  2004 ASCRS and ESCRS

T he design of intraocular lenses (IOLs) should be


based on the properties of the human ocular lens,
especially in the transmission properties of the IOL.
human lens, since the latter yellows with age while IOLs
in current use are colorless.2,9–13 The color change in the
natural lens is likely attributable to oxidation products of
Notably, the human crystalline lens absorbs most ultra- tryptophan (n-formyl-kynurenine) and to glycosylation
violet light between 300 nm and 400 nm.1 Thus, to of lens proteins.14 It results in a progressive increase
provide the same protection to the retina afforded by in absorbance within the blue range of the visible
the natural lens, the use of ultraviolet light-absorbing spectrum.2,9
IOLs became the standard of practice once these lenses Filtering the shorter wavelengths of the visible spec-
were developed.2–8 However, the transmission properties trum is particularly significant because it is also this
of most IOLs are not comparable to those of the natural portion of the spectrum that produces photochemical
damage to the retinal pigment epithelium (RPE).15–17
It is now generally accepted that at least 1 of the intracel-
Accepted for publication January 27, 2004.
lular chromophores responsible for the blue light sensi-
Reprint requests to Janet R. Sparrow, PhD, Department of Ophthalmol-
ogy, Columbia University, 630 West 128th Street, New York, New tivity of RPE cells18,19 is the lipofuscin constituent
York 10032, USA. email: jrs88@columbia.edu. A2E.20–22 This fluorophore is unique to RPE cells and
 2004 ASCRS and ESCRS 0886-3350/04/$–see front matter
Published by Elsevier Inc. doi:10.1016/j.jcrs.2004.01.031
LABORATORY SCIENCE: BLUE LIGHT-ABSORBING IOL

has been shown to accumulate in human RPE cells CeeOn威 Edge (911A) (silicone, 20.0 D, 6.0 mm optic
throughout the lifetime of an individual, with levels diameter).
being highest in the aged eye.21 Thus, replacement of
a senile cataractous crystalline lens with a colorless IOL A2E Accumulation in Culture
may leave the RPE vulnerable at an age when its content Human RPE cells (ARPE-19, American Type Culture
of blue light sensitive A2E is already high. Collection), which are devoid of endogenous A2E,35 were
grown in 8-well, plastic chamber slides (Laboratory-Tek,
The RPE fluorophore A2E is maximally excited
Nunc), as described.35 Once confluent, the cells were allowed
by light in the blue region of the spectrum.18 When to accumulate synthesized A2E21 from a 20 ␮M concentration
irradiated, A2E generates singlet oxygen that proceeds to added to the medium. With this protocol, A2E accumulates
add to carbon–carbon double bonds of A2E to generate in the lysosomal compartment of the cells to levels that are
highly reactive epoxides (A2E-epoxides) along the side- comparable to amounts present in vivo.18,35
arms of the molecule.23, 24 The cellular injury induced by
the illumination of A2E-laden RPE includes oxidative Illumination and Placement of the IOL
DNA base changes,25,26 and it is likely that as electro- Immediately before illumination, the culture medium
philes that can readily react with many cellular mole- was replaced with phosphate-buffered saline containing cal-
cules, A2E-epoxides may account for much of the cium, magnesium, and glucose. For quantitative measure-
cellular damage accrued.25 The photochemical events ments of light-induced cell death, the cells were exposed to
provoked by the irradiation of A2E-laden RPE ulti- blue light (430 nm ⫾ 30 [SD], 8 mW/cm2), green light
(550 ⫾ 10 nm, 8 mW/cm2), or white light (246 mW/cm2)
mately result in the initiation of a cell death program.
over a 0.8 mm ⫻ 8.5 mm field. The light was delivered
The sensitivity to blue light conferred by the lipofuscin
from a tungsten halogen source for 20 minutes, and power
fluorophore A2E may explain why atrophic age-related was measured with a Newport optical power meter (model
macular degeneration (AMD) has been linked to both 840). The spectral range of the lamp was 390 to 750 nm,
RPE lipofuscin27–33 and cumulative light exposure.34 with a lower output at the shorter wavelengths.
Moreover, the loss of RPE cells in atrophic AMD is a The IOL to be tested was applied to the undersurface
critical event as it leads to photoreceptor cell degeneration. of the culture well, where it remained attached, unaided. It
Given that the attenuation of blue light afforded was centered over the light path.
by the yellowed senescent lens may defend lipofuscin- To obtain a visual representation of IOL protection,
filled RPE cells against blue light damage, we are in- 1.0 mm diameter disks were cut from the center of the IOL
using a trephine blade (Katena Products, Inc.). The IOL
terested in efforts being made to develop IOLs that
disks were positioned on the undersurface of the well, which
replicate the transmission characteristics of the aging
was then irradiated (430 ⫾ 30 nm, 16 mW/cm2) from below.
crystalline lens. In this study, we constructed a cell
culture system that allowed us to compare several IOLs
Detection of Nonviable Cells
as to their ability to protect A2E-laden RPE from blue
light damage. In this cell culture system, A2E accumu- The nuclei of dead RPE cells were labeled with a mem-
brane impermeant dye (Dead Red, Molecular Probes), and
lates in the lysosomal compartment of a human RPE the nuclei of all cells with 4⬘,6⬘-diamino-2-phenylindole
cell line, as it does in RPE of the eye.35 Moreover, the (DAPI), as reported.25,26 Blue light-illuminated A2E-con-
levels of A2E are comparable to the level occurring in taining RPE labeled in this way are undergoing an apoptotic
vivo.18,35 This model also allows us to study RPE cells form of cell death.36 Digital images (5 fields per illumination
with and without intracellular deposits of A2E. zone) were obtained using a Zeiss Axioplan II microscope
with Axiocam camera and KS400 image processing software.
Subsequently, Dead Red and DAPI-stained nuclei were
Materials and Methods counted. Dead cells were quantified as a percentage of the
The IOLs studied were the Alcon AcrySof威 Natural total number of cells in a field. Means are based on 3
(SN60AT) (acrylic, 20.0 diopters [D], 6.0 mm optic diame- experiments.
ter) and AcrySof (SA60AT) (acrylic, 20.0 D, 6.0 mm optic Statistical analysis was by an analysis of variance followed
diameter); the AMO ClariFlex威 (CLRFLXB) (silicone, by the Newman-Keul multiple comparison test (Prism,
20.0 D, 6.0 mm optic diameter) and Sensar威 (AR40e) GraphPad Software). A P value of 0.05 or less was consid-
(acrylic, 20.0 D, 6.0 mm optic diameter); and the Pharmacia ered significant.

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LABORATORY SCIENCE: BLUE LIGHT-ABSORBING IOL

that did not contain A2E (no A2E, no IOL) remained


viable, while blue light-illuminated (430 nm peak with
a bandwidth of 60 nm, 8 mW/cm2) RPE that had
previously accumulated A2E underwent marked cell
death. In the present experiments, 41.1% ⫾ 4.1% (3
experiments) of the A2E-laden cells in a field of illumi-
nation became nonviable after blue light exposure in
the absence of an IOL (Figure 1). When the yellow-
tinted AcrySof Natural IOL was placed in the center
of the light path, transmission of the 430 nm light
was reduced by approximately 50% and cell death was
reduced by 80% (P⬍.001) compared to irradiation in
the absence of an IOL (Figure 1). When the AcrySof,
Sensar, ClariFlex, or CeeOn Edge IOLs were in place,
Figure 1. (Sparrow) Quantitation of nonviable RPE cells after A2E the frequency of nonviable cells was not different
accumulation and blue light illumination (430 nm) with and without (P⬎.05) than cell death in the absence of an IOL. The
an IOL placed in the light path. The percentage of nonviable cells
modest but consistent declines in cell death that were
was determined by labeling all nuclei with DAPI and the nuclei of
nonviable cells with a membrane-impermeable dye. The data were detected with these IOLs (Figure 1) were undoubtedly
normalized to the percentage of nonviable cells in the absence of an due to the small reductions in light transmission (⬃5%)
IOL. Values are mean ⫾ SEM. that were measured.
The ability of the AcrySof Natural IOL to attenuate
blue light-mediated cell death was evident when fields
of irradiated cells were imaged. In the absence of an IOL,
the nuclei of nonviable cells were uniformly distributed
across the illuminated field (Figure 2). Conversely,
placement of the blue light-absorbing AcrySof Natural
IOL (a 1.0 mm disk) within the light path spared a
circular zone of cells, the diameter of this area correspond-
ing to the diameter of the IOL disc. Within this sector,
all nuclei were labeled with DAPI but labeled nuclei
of nonviable cells were only occasionally observed.
With the use of wide-band white light (390 to
750 nm; 246 mW/cm2) that included all wavelengths
toward which A2E exhibits sensitivity, the death of
A2E-laden RPE reached a frequency of 35% (35.4% ⫾
Figure 2. (Sparrow) A blue light-absorbing IOL protects A2E- 2.7% of the cells in an illumination zone; 3 experi-
laden RPE from cell death due to 430 nm irradiation. The nuclei of ments). In the presence of the blue light-absorbing Acry-
nonviable RPE were labeled with a membrane impermeable dye (left), Sof Natural IOL, cell death from white light was
and all nuclei were stained with DAPI (right). The left and right panels
reduced by 82% (P⬍.001) compared to that without
are corresponding fields of illuminated A2E-laden RPE. In the absence
of an IOL in the light path (top), the entire field is covered with nonviable an IOL (Figure 3). In comparison, the AcrySof IOL
cells. Placement of a blue light-absorbing IOL (1.0 mm diameter disk; diminished the incidence of nonviable cells by 22%
AcrySof Natural) in the light path reduced the number of nonviable (P⬍.05); with the Sensar, ClariFlex, and CeeOn Edge
cells in a circular zone corresponding to the area covered by the IOL
IOLs, cell death was decreased by 37% to 39% (P⬍
(bottom, dotted circle).
.01). Again, the decreases observed with the colorless
IOLs were likely related to the reductions in spectral
Results transmittance associated with all IOLs.
Using a fluorescence assay that labels the nuclei of When compared with blue and white light, green
nonviable cells, it was observed that illuminated RPE light (550 ⫾ 20 nm) delivered at an intensity that was

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Figure 3. (Sparrow) Quantitation of nonviable cells after white light Figure 4. (Sparrow) Quantitation of nonviable cells after green light
(390 to 750 nm) illumination of A2E-laden RPE with and without the illumination (550 nm) of A2E-laden RPE with and without the indicated
indicated IOL placed in the light path. The percentage of nonviable IOL placed in the light path. Values for percentage of nonviable cells
cells was normalized to values obtained in the absence of an IOL. were normalized to values obtained in the absence of an IOL. Means ⫾
Values are mean ⫾ SEM. SEM are presented.

the same as that of the blue light (8 mW/cm2) induced with the other IOLs were probably due to IOL-associ-
the death of only a small number of A2E-laden RPE ated nonspecific decreases (5% to 10%) in light trans-
cells (2.9% ⫾ 0.6%; 3 experiments). With green light mission that have been reported.2,7,8,37
irradiation, all IOLs reduced the death of RPE that had The AcrySof Natural IOL did not completely abol-
accumulated A2E (Figure 4). With the AcrySof Natural, ish cell death in this model. However, this is not surpris-
the number of nonviable cells was decreased by 78% ing since by design, this IOL does not block all blue
(P⬍.01) compared to cell death without an IOL; while light. Rather, the yellow tint of the lens is intended to
cell death declined by 33% with the AcrySof (P⬎.05) confer a spectral transmission similar to that of the
and by 52% to 57% (P⬍.05) with the Sensar, ClariFlex, crystalline lens of an adult.1 That A2E-containing RPE
and CeeOn Edge IOLs. are less sensitive than to green light is to be expected:
A2E has an excitation maximum of approximately
430 nm with the emission elicited at 550 nm being less
Discussion than 5% of that elicited at 430 nm.18
The study shows that by absorbing predominantly Ham et al.15 were the first investigators to demon-
in the blue region of the spectrum, the AcrySof Natural strate that retinal injury from wavelengths that peak
IOL shields RPE cells that have accumulated the lipofus- around 425 nm is initiated by photochemical processes
cin fluorophore A2E from the damaging effects of light. in the RPE. The action spectrum of this damage corre-
The protection afforded by the AcrySof Natural IOL sponds to the absorption spectrum of the aging fluo-
was pronounced for wide-band white light just as it rophore A2E,18 and there is now an abundance of
was for narrow-band blue light. The reduction in cell experimental evidence that A2E is a sensitizing molecule
death afforded by this blue light-filtering IOL was that can mediate light injury to RPE, leading to RPE
greater in magnitude than would be expected from the atrophy.18,23,25,26,36 Since lipofuscin fluorophores, includ-
decline in transmitted blue light. That a reduction in ing A2E, accumulate with age, the natural yellowing
blue light transmission of approximately 50% resulted of the aging human lens is fortunate as it may dampen
in an 80% decline in cell death probably occurred be- the damaging potential of short wavelength blue light.
cause blue light levels were brought below the threshold For some time, it has been postulated that cumula-
for lethal damage for a significant proportion of the tive photochemical damage from lifetime exposures to
cells. Small but consistent declines in cell death observed light are a cause of AMD,38 although the results of

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epidemiological studies are inconclusive.39,40 Neverthe- 5. Lawrence HM, Reynolds TR. Erythropsial phototoxi-
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Sci 1997; 38:478–486 From the Department of Ophthalmology, Columbia University, New
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None of the authors has a financial or proprietary interest in any
tional zone of geographic atrophy of the retinal pigment product mentioned.
epithelium associated with age-related macular degenera-
tion. Graefes Arch Clin Exp Ophthalmol 1999; 237: Presented at the XXI Congress of the European Society of Cataract and
145–152 Refractive Surgeons, Munich, Germany, September 2003.
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related macular degeneration. Invest Ophthalmol Vis Sci restricted funds from Research to Prevent Blindness, New York, New
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